Helminth infections are responsible for severe health problems worldwide, in both human and animal populations. It is estimated that 2-3 billion people, mainly from rural and impoverished regions of the world suffer from disease due to parasitic nematodes; however, drug development and discovery efforts lack commitment to combat human infection due to the poor commercial market. In contrast, there are robust animal health discovery efforts aimed at eradicating infection in valuable domesticated livestock herds. Thus, animal health programs are often relied on to provide drug candidates for human therapeutics. Extensively tested in animals, the cost and development risk of human drugs originating from animal health are reduced. While human drug candidates still need to surpass stringent safety standards, an equal challenge is minimizing cost of goods to enable the infected population access to potent, low cost therapeutics. Accordingly, paraherquamide represents a new class of anti-helminth antibiotics that possesses broad-spectrum anti-nematodal activity and is a precursor to a recently launched animal health anti-parasitic agent 2- desoxoparaherquamide (Derquantel(tm)). Currently paraherquamide is manufactured through fermentation of the fungal strain P. simplicissimum. To maximize yields, the drug manufacturer has embarked on strain improvement programs to randomly evolve high producing mutants; however, this program has failed to achieve the desired output. Here, Alluvium proposes a modernized, genomics-driven strain improvement strategy aimed at maximizing paraherquamide production in fermentation. Specifically, Alluvium's approach is guided by the DNA sequence of the fungal genome in which key regulatory and/or biosynthetic genes responsible for paraherquamide production have been identified. Rational genetic engineering strategies, such as gene deletion or gene amplification, will be applied to key genes in efforts to rationally generate a high producing mutant phenotype. In this Phase I proposal, work is focused on rationally manipulating three genes that are predicted to regulate, both positively and negatively paraherquamide biosynthesis. In addition, Alluvium proposes to replicate the entire paraherquamide biosynthetic gene cluster within the fungal chromosome in order to boost production of anti-helminthic natural product. Following success in these initial studies, Phase II work will focus on engineering further efficiencies in paraherquamide production, including developing biocatalytic methods capable of converting paraherquamide to 2-desoxoparaherquamide as well as biotransformation tools that can be employed to generate synthetically challenging structural analogs. In sum, the strain engineering technology under development at Alluvium will result in an optimized paraherquamide manufacturing process that will lower the cost of goods required to access this commercially important anti- helminthic therapeutic, thereby enabling the potential development of a much-needed human therapeutic. PUBLIC HEALTH RELEVANCE: Helminths, especially parasitic nematodes, cause severe health problems in both humans and domesticated animals. Approximately 2-3 billion people in rural and impoverished regions are infected; however, affordable and effective treatment options are scarce. The proposed work seeks to improve upon the production of the promising anti-helminthic animal health chemotherapeutic agent, paraherquamide, so it can be generated in a more cost-effective and efficient manner, thereby stimulating further development activities for potential human treatment.